B1 cells represent a distinct lymphocyte lineage and a fundamental component of the immune system. B1 cells are associated with autoimmunity and malignancy, and are responsible for the production of natural immunoglobulin that fulfills a critical anti-bacterial function. PD-L2 is a B7 family member previously reported to be expressed primarily by macrophages and dendritic cells but now shown by work from this laboratory to be expressed by B1 cells. Not only do B1 cells express PD-L2, but PD-L2 expression marks B1 cells that manifest enhanced repertoire skewing, increased autoantibody production, amplified replication history, and augmented antigen presentation, in comparison to PD-L2 nonexpressing B1 cells and to B2 cells. These characteristics have long been associated with B1 cells in general, but this new work indicates that these features predominantly segregate to a B1 cell subset defined by PD-L2 expression. The long term objective of this proposal, and of previous projects in this sequence, is to understand how B1 cells get to be the way they are, and what is the role and function of B1 cells within the immune system. PD-L2 expression by a subset of B1 cells provides a new and important tool to address these issues. Because PD-L2+ B1 cells preferentially express characteristics normally associated with the B1 cell lineage, these findings infer a new paradigm wherein PD-L2 plays a role in producing those characteristics, or is otherwise associated with them. Conversely, because PD-L2 expression is limited in scope and has not previously been reported by lymphocytes, these findings infer a new paradigm wherein a fraction of B1 cells participate in PD-L2-mediated effects. The specific aims of this proposal are to: 1) Determine the regulatory elements that control PD-L2 expression in B1 cells through an analysis of promoter sequences and binding factors; 2) Determine the developmental progression of PD-L2-expressing B1 cells through the study of phenotypically defined early stages in B cell differentiation; and, 3) Determine the role of PD-L2 in specifying the unique features of PD-L2- expressing B1 cells through manipulation of PD-L2 expression in mature and developing B1 cells. The results of this study are expected to provide new information regarding PD-L2 gene regulation in B1 cells, regarding PD-L2 expression during B cell ontogeny, and regarding the mechanism by which PD-L2 expression correlates with several characteristics previously attributed to B1 cells in general, particularly autoantibody production. These are fundamental issues that relate to B1 cell behavior and activity, and PD-L2 expression and function, through the study of which much new will be learned regarding the role and place of B1 cells within the immune system. Elucidation of these points is likely to provide new targets and strategies to ameliorate the progression of autoimmune dyscrasias, to influence the course of malignant diseases, and to assist in enhancing immunity in normal and immune-deficient patients. PUBLIC HEALTH RELEVANCE: B1 cells are a subpopulation of B lymphocytes, the immune cells that produce antibody. B1 cells behave differently from other B cells in many respects-they are especially valuable because they produce disease- fighting antibody without the need for vaccination, but they are problematical because they are involved in initiating or aggravating autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosis. The work described in this proposal builds on the discovery that some B1 cells express a surface molecule never before known to be expressed by lymphocytes, and that the B1 cells marked by this molecule have special properties in terms of making antibody, including autoimmune autoantibody, and other characteristics. The hypothesis that this molecule actually causes the special properties of B1 cells that express it will be tested. Ideally, this research will provide information useful for manipulating B1 cell activity-downward, to improve the condition of patients afflicted with autoimmune diseases, and upward, to improve the condition of patients afflicted with immune deficiency states.